Surgical resection or liver transplantation remain the only curative options for patients with hepatocellular carcinoma (HCC). However, fewer than 20% of patients with HCC are candidates for resection. Transarterial chemoembolization (TACE) is an endovascular locoregional embolotherapy that involves hepatic artery embolization with intra-arterial infusion of a chemotherapeutic agent and is considered the standard of care for treating unresectable HCC in the remaining 80% of patients with this disease. While TACE has a proven survival benefit, local recurrence is common, and long-term survival rates are poor. Moreover, only 44% of treated HCCs demonstrate extensive necrosis on pathology following TACE, indicating that tumor cells develop an adaptive metabolic stress response (MSR) enabling their survival under TACE-induced nutrient and oxygen deprivation. In preliminary studies, we have demonstrated that HCC cells may be pre-programmed to survive TACE-induced ischemia through enhanced function of the MSR, including autophagy and hypoxia-inducible factors (HIFs). Moreover, TACE-induced ischemia results in quiescence in surviving HCC cells and further activation of the MSR. These data demonstrate that TACE offers a unique opportunity to constrain metabolic phenotypes in order to generate targetable dependencies in HCC. The proposed project will build on this prior work to: 1) further characterize the role of targetable MSR pathways in enabling HCC cell survival under TACE-like ischemia and 2) validate a synergistic therapeutic strategy that targets this MSR dependence in order to define a novel, and more effective, approach to TACE. Gene editing will be used to examine the roles of fructose bisphosphatase 1 and urea cycle enzymes in influencing the basal activity of HIFs and autophagy, respectively in HCC cells. The dependence of HCC cell survival on each of the MSR pathways individually and in combination will be determined through gene editing and pharmacologic inhibition. Our recently developed, unique autochthonous rat model of HCC and TACE will be utilized to assess the TACE-induced essentiality of each MSR pathway in vivo using pharmacological inhibitors of the MSR. We hypothesize that the induction of quiescence in HCC cells surviving TACE-induced ischemia renders them dependent on MSR pathways which can be targeted to potentiate the cytotoxic effects of TACE. To test this hypothesis the proposed project will pursue three aims: (1) to define genetic alterations that contribute to molecular and cellular stress response pathway activation in HCC in vitro and in vivo; (2) to functionally demonstrate the dependence of HCC cells surviving severe ischemia on induction of the MSR in vitro; and (3) to determine the efficacy of potentiating TACE-induced ischemia by individual and simultaneous pharmacologic targeting of the MSR in vivo. Importantly, the proposed project emphasizes a translational approach, so that the achievement of these aims will directly inform and influence the treatment of patients with unresectable HCC, an incurable disease.